Domain Coupling in Allosteric Regulation of SthK Measured Using Time-Resolved Transition Metal Ion FRET

Reviewer #1 (Public review):

Summary:

This useful work extends a prior study from the authors to observe distance changes within the CNBD domains of a full-length CNG channel based on changes in single photon lifetimes due to tmFRET between a metal at an introduced chelator site and a fluorescent non-canonical amino acid at another site. The data are excellent and convincingly support the authors' conclusions. The methodology is of general use for other proteins. The authors also show that coupling of the CNBDs to the rest of the channel stabilizes the CNBDs in their active state, relative to an isolated CNBD construct.

Strengths:

The manuscript is very well written and clear.

Reviewer #2 (Public review):

The manuscript "Domain Coupling in Allosteric Regulation of SthK Measured Using Time-Resolved Transition Metal Ion FRET" by Eggan et al. investigates the energetics of conformational transitions in the cyclic nucleotide-gated (CNG) channel SthK. This lab pioneered transition metal FRET (tmFRET), which has previously provided detailed insights into ion channel conformational changes. Here, the authors analyze tmFRET fluorescence lifetime measurements in the time domain, yielding detailed insights into conformational transitions within the cyclic nucleotide binding domains (CNBDs) of the channel. The integration of tmFRET with time-correlated single-photon counting (TCSPC) represents an advancement of this technique.

The results summarize known conformational transitions of the C-helix and provide distance distributions that agree with predicted values based on available structures. The authors first validated their TCSPC approach using the isolated CNBD construct previously employed for similar experiments. They then study the more complex full-length SthK channel protein. The findings agree with earlier results from this group, demonstrating that the C-helix is more mobile in the closed state than static structures reflect. Upon adding the activating ligand cAMP, the C-helix moves closer to the bound ligand, as indicated by a reduced fluorescence lifetime, suggesting a shorter distance between the donor and acceptor. The observed effects depend on the cAMP concentration, with affinities comparable to functional measurements. Interestingly, a substantial amount of CNBDs appear to be in the activated state even in the absence of cAMP (Figure 6E and F, fA2 ~ 0.4).

This may be attributed to cooperativity among the CNBDs, which the authors could elaborate on further. In this context, the major limitation of this study is that distance distributions are observed only in one domain. While inter-subunit FRET is detected and accounted for, the results focus exclusively on movements within one domain. Thus, the resulting energetic considerations must be assessed with caution. In the absence of the activator, the closed state is favored, while the presence of cAMP favors the open state. This quantifies the standard assumption; otherwise, an activator would not effectively activate the channel. However, the numerical values of approximately 3 kcal/mol are limited by the fact that only one domain is observed in the experiment, and only one distance (C- helix relative to the CNBD) is probed. Additional conformational changes leading to pore opening (including rotation and upward movement of the CNBD, and radial dilation of the tetrameric assembly) are not captured by the current experiments. These limitations should be taken into account when interpreting the results.

Reviewer #3 (Public review):

Summary:

This is a lucidly written manuscript describing the use of transition-metal FRET to assess distance changes during functional conformational changes in a CNG channel. The experiments were performed on an isolated C-terminal nucleotide binding domain (CNBD) and on a purified full-length channel, with FRET partners placed at two positions in the CNBD.

Strengths:

The data and quantitative analysis are exemplary, and they provide a roadmap for use of this powerful approach in other proteins.

Weaknesses/Comments:

A ~3x lower Kd for nucleotide is seen for the detergent-solubilized full-length channel, compared to electrophysiological experiments. This is worth a comment in the Discussion, particularly in the context of the effect of the pore domain on the CNBD energetics.